치은 각화상피세포와 섬유아세포를 이용한 삼차원적 배양시 중층화 동안의 변화

정태흡,현하나,김윤상,김은철,유형근,신형식,Jung, Tae-Heup,Hyun, Ha-Na,Kim, Yun-Sang,Kim, Eun-Cheol,You, Hyung-Keun,Shin, Hyung-Shik 대한치주과학회 2002 Journal of Periodontal & Implant Science Vol.32 No.1

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.

In vitro evaluation method to monitor pathogenic sensitive skin by co-culture of keratinocytes and neuronal cell line

( Sun Mee Shin ),( Eun Hye Hong ),( Soo Hyun Jeong ),( Eun Joo Park ),( Kwang Joong Kim ),( Kwang Ho Kim ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Background: Keratinocytes have recently known to participate in sensory transduction by release of neuroactive molecules which bind to intra-epidermal free nerve endings (FNEs) and modulate nociception. Although reciprocal interactions between keratinocytes and FNEs via soluble mediators are well established, little is known about physical contacts between keratinocytes and sensory neurons. Objectives: We developed for the first time the co-culture of human primary keratinocyte with differentiated SH-SY5Y to reproduce direct interactions in vitro. Methods: We investigated the morphological and functional characteristics of differentiated SH-SY5Y neuronal cell line co-cultured with primary keratinocyte and analyzed the influence of keratinocytes. Results: SH-SY5Y cells survived well in keratinocyte co-culture condition. When SH-SY5Y cells were co-cultured with keratinocytes, they had no significant influence on axonal development. The neuropeptide Substance P (SP) which is released after a cytosolic calcium influx and modulates immediate skin hypersensitivity was also released well after capsaicin treatment in co-cultured condition. Conclusion: This co-culture model in which keratinocytes and neurons may be a useful in vitro alternative for studying and characterizing the close communication between keratinocytes and sensory neurons.

진주종 각질형성세포와 섬유아세포의 삼차원적 분리 공배양

김석천,홍남표,추재학,박수홍,홍석찬,차창일 대한이비인후과학회 2000 대한이비인후과학회지 두경부외과학 Vol.43 No.12

동종유래각질세포(Cultured Allogenic Keratinocytes, Kaloderm^®)를 이용한 부분층 피부 결손의 치료

서상원,장충현,조민수,홍윤기,전세화,Seo, Sang Won,Chang, Choong Hyun,Cho, Min Su,Hong, Yoon Gi,Jeon, Sae Wha 대한외상학회 2007 大韓外傷學會誌 Vol.20 No.1

Purpose: Grafting with autograft skin remains the most effective method for treating skin defects. When insufficient donor sites are present or patients are afraid of the operation, a skin graft is impossible. Cultured allogenic keratinocytes speed wound healing by providing cover and by producing growth factors and extracellular matrix protein. We report an application of cultured allogenic keratinocytes ($Kaloderm^{(R)}$, Tegoscience, Seoul, Korea) in the treatment of an acute partial thickness skin defect. Methods: From March 2005 to January 2006, 20 patients with a partial thickness skin defect were treated with cultured allogenic keratinocytes. The wound was covered with a sheet of cultured allogenic keratinocytes and ointment with $Bactigras^{(R)}$ gauze. The wound was inspected every two or three days. We regarded completion of epithelialization as wound healing. Results: The mean period between time of injury and time of $Kaloderm^{(R)}$ application was 7.5 days. The time taken from application of $Kaloderm^{(R)}$ to complete closure of the wounds was 7.2 days. Conclusion: In view of the favorable outcome, cultured allogenic keratinocytes are safe and effective biologic dressing materials for use in the treatment of open wounds.

동종유래각질세포(Cultured Allogenic Keratinocytes, Kaloderm(R))를 이용한 부분층 피부 결손의 치료

서상원 ( Sang Won Seo ),장충현 ( Choong Hyun Chang ),조민수 ( Min Su Cho ),홍윤기 ( Yoon Gi Hong ),전세학 ( Sae Wha Jeon ) 대한외상학회 2007 大韓外傷學會誌 Vol.20 No.1

Purpose: Grafting with autograft skin remains the most effective method for treating skin defects. When insufficient donor sites are present or patients are afraid of the operation, a skin graft is impossible. Cultured allogenic keratinocytes speed wound healing by providing cover and by producing growth factors and extracellular matrix protein. We report an application of cultured allogenic keratinocytes (Kaloderm(R), Tegoscience, Seoul, Korea) in the treatment of an acute partial thickness skin defect. Methods: From March 2005 to January 2006, 20 patients with a partial thickness skin defect were treated with cultured allogenic keratinocytes. The wound was covered with a sheet of cultured allogenic keratinocytes and ointment with Bactigras(R) gauze. The wound was inspected every two or three days. We regarded completion of epithelialization as wound healing. Results: The mean period between time of injury and time of Kaloderm(R) application was 7.5 days. The time taken from application of Kaloderm(R) to complete closure of the wounds was 7.2 days. Conclusion: In view of the favorable outcome, cultured allogenic keratinocytes are safe and effective biologic dressing materials for use in the treatment of open wounds. (J Korean Soc Traumatol 2007;20:1-5)

정상 및 HPV 불멸화 인간 구강상피의 삼차원적 배양

김성현,이선경,이화정,황영수,박대열,장현주,허용,김은철 대한구강악안면병리학회 2003 대한구강악안면병리학회지 Vol.27 No.4

Epithelial-mesenchymal interaction is well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular micro-environment which provide a epithelial-mesenchymal interaction. The aims of this study were to develop and evaluate the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted human normal oral kertinocyte(NHOK) and immortalized human oral keratinocytes(IHOK) by histological and immunohistochemical analysis. The results were as follows; 1. Best condition of three dimensionally reconstituted IHOK & HaCaT cells are 14 days air-exposure cultivation, 3 days of submerged state, and dermal equivalent consisting type I collagen and IGF cells. 2. In comparison to IHOK, there was better preservation of the overall epidermal strucutures in oral cracioma cells (HN30) & HaCaT cells by organotypic cultures. But orgnaotypic co-culture of the normal keratinocyte showed the thinnest epithelial layer formation. 3. PCNA was detected primarily in the basal layer of normal mucosa and NHOK, whereas was shown throught the epithellium except surface layer of IHOK cells, and it's expression was similar to that of CIS of biopsied patient's tissue 4. Involucrin is expressed in the upper layer of oral mucosa and NHOK raft, but staining for involucrin was induced in the IHOK rafts indicating differentiation is incomplete, and the staining pattern in the IHOK raft was not uniform. 5. Normal oral keratinocyte raft showed weak immunostaining for p53, and p53 expression of IHOK raft increased rahter than in NHOK. In organotypic cultures of normal cells and IHOK, p53 expression was restricted to the proliferative part of epithelium. This is consistent with expression pattern in biopsy specimens of the normal and CIS tissue. 6. In artifically reconstructed NHOK, the pattern of keratin staining showed both similarity and differences from that of intact normal mucosa. An obvious difference was increased expression of CK10 & CK19, and decreased expression of CK6 in a reconstructed NHOK rather than in normal mucosa, and similar expression was in CK4 and CK16. 7. CK19 & CK16 were strongly positive in HPV immortlaized keratinocyte rafts rahter than in NHOK, an indicator of premalignant or malignant changes, while CK10 & CK6 were decreased, and organotypic cultures of IHOK express was similar keratin expression as epithelial dysplasia or CIS tissue. These results suggest that three-dimensional organotypic co-culture of normal oral & immortalized keratinocytes with dermal equivalent consisting type I collagen and fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. Thus this organotypic system can be used for studing mechanism of epitehlial-mesenchymal interaction in particular regulating epidermal diffenentiation and morphogenes

피부 기관 배양에서 칼슘이 표피에 미치는 효과

최대경 ( Dae Kyoung Choi ),이경문 ( Kyung Moon Lee ),김대훈 ( Dae Hun Kim ),이영 ( Young Lee ),손경철 ( Kyung Cheol Sohn ),김창덕 ( Chang Deok Kim ),서영준 ( Young Joon Seo ),이증훈 ( Jeung Hoon Lee ) 대한피부과학회 2010 대한피부과학회지 Vol.48 No.5

Background: Calcium plays a role in the proliferation and differentiation of keratinocytes. In a normal situation, the calcium concentration forms a gradient across the epidermal layers. Calcium is sparse in the basal layer and spinous layer. Skin organ culture is a useful model for conducting research on various aspects of skin biology. Skin organ culture systems are used for defining factors that affect homeostasis when elucidating the modulatory effects of biologic response modifiers, drugs and physical agents on the skin and also when studying complex aspects of cutaneous biology in normal and diseased skin. Objective: In this study, we investigated the effects of extracellular calcium on the epidermis in a skin organ culture. Methods: We compared the skin organ culture patterns under various culture conditions (calcium 0.1, 0.7, 1.4 and 2.0 mM). Results: H&E staining showed different phenotypes according to the calcium concentration and IHC also showed different phenotyes compared to that of keratin 10, involucrin, filaggrin, loricrin and PCNA. Conclusion: As a result, we concluded that the calcium gradient is also an important factor in skin organ culture to maintain the vivo-like environment and the appropriate calcium concentration is 1.4 mM. (Korean J Dermatol 2010;48(5):373~379)

화상치료에 있어서 동종유래표피세포의 유용성

윤신혁,심정수,정재민,박대환,송철홍 대한성형외과학회 2008 Archives of Plastic Surgery Vol.35 No.4

Purpose: When choosing dressing method to treat skin defect by second degree or higher burn, we have to consider method of rapid epithelization and minimization of pain during the treatment. In this study, we used biologic dressing with cultured allogenic keratinocytes for skin defect due to burn. We followed up the degree of epithelization, the degree of pain, and patient satisfaction. Methods: From June 2003 to June 2006, among the patients with skin defect due to burn, 31 cases with second degree burn(moderate to severe) were selected and biological dressing with cultured allogenic keratinocytes were done. 21 cases did not use cultured allogenic keratinocytes. Most of the patients had second degree burn. We applied cultured allogenic keratinocyte by Kaloderm. For wounds that were not deep enough to effect the dermis, escharectomy was done before applying Kaloderm. After the operation, moist wound site was maintained by dressing with saline gauze for 5-7 days. We compared the condition of the wound site before and after applying Keloderm by grading epithelization by standardized percentage scoring scale(1-5), and degree of pain and patient satisfaction by visual analogue scale(0-10). Received March 31, 2008 Revised April 23, 2008 Accepted May 22, 2008 Address Correspondence: Jeong Su Shim, M.D., Department of Plastic and Reconstructive Surgery, College of Medicine, Catholic University of Daegu, 3056-6 Daemyung 4-dong Nam-gu, Daegu 705-718, Korea. Tel: 053)650-4578/Fax: 053)650-4584/E-mail: 21csue@hanmail.net Results: When cultured allogenic keratinocytes were applied for the same period of time, the mean score of epithelization were 3.29±0.529(mean±S.D.). Without the application, the mean score of epithelization were 2.86±0.655(mean±S.D.). The degree of pain was 7.71 ±1.419(mean±S.D.) and 2.35±0.950(mean±S.D.) before and after the application, respectively. The patients' satisfaction score was 6.45±0.850(mean± S.D.) and 8.45±0.961(mean±S.D.) before and after the application, respectively. Conclusion: Applying biological dressing with cultured allogenic keratinocyte to skin defect due to second degree burn showed satisfactory results in the degree of the epithelization, degree of pain and patients' satisfaction.

FCT 2-5 A case of stable vitiligo treated with non-cultured keratinocyte-melanocyte transplantation

( Yu Seok Jung ),( Hanna Lee ),( Ji Hae Lee ),( Gyong Moon Kim ),( Jung Min Bae ) 대한피부과학회 2016 대한피부과학회 학술발표대회집 Vol.68 No.2

Background: Surgical interventions for stable vitiligo include cellular and tissue grafting. Cellular grafting includes transplantation of non-cultured keratinocyte- melanocyte suspension and transplantation of cultured pure melanocytes, whereas tissue grafting includes punch, split-thickness and blister roof grafting. Objectives: To demonstrate the efficacy and safety of non-cultured keratinocyte-melanocyte transplantation in stable vitiligo. Methods: A 14-year old female patient presented with large depigmented patch on her lower leg. Intact epidermis was harvested from the normal skin of inner thigh using suction blister technique, and then incubated with trypsin-ethylene-diamine tetra-acetic acid solution at 37 °C for 45 minutes to separate epidermal cells. The cell suspension was filtered through a 70 μm cell strainer. Finally, the cell suspension was centrifuged for 10 min at 1500 rpm. to obtain a cell pellet, and applied to denuded recipient site. Then, the lesion was dressed with vaseline gauze for 7 days, and the narrow-band ultraviolet B therapy was resumed after 21 days. Evaluation of repigmentation was performed 12-week post transplantation. Results: At the 12-week follow-up visit, more than 95% of repigmentation was obtained. No adverse events including infection or visible scarring at any donor or recipient site were noted. Conclusion: Non-cultured keratinocyte-melanocyte transplantation appeared to be safe and effective in the treatment of stable vitiligo.

PUVA가 배양 각질형성세포의 성장과 배양 멜라닌세포의 성장 , 멜라닌양 , 수지상돌기 수 및 길이에 미치는 영향

전일선 ( Il Sun Jun ),박재경 ( Jae Kyung Park ),허충림 ( Choong Rim Haw ),이무형 ( Mu Hyoung Lee ) 대한피부과학회 1995 大韓皮膚科學會誌 Vol.33 No.5

Background : The combination of psoralen and UVA (PUVA) as a model of photochemotherap, has been used in a wicle variety of cutaneous disorders such as psoriasis, vitiligo, mycosis fungoides and atopic dermatitis. The mechanism of PUVA of psoriasis is based on the fact. that PUVA causes photoconjugation of psoralens to DNA and a subsequent suppression of mitosis, DNA synthesis, and keratinocyte proliferation. Although PUVA apparently inhibits keratinocytie proliferation and is effective therefore in the treatment of psoriasis, PUVA increases pigmentations by stimulating melanocyte proliferation and melanin synthesis in vivo. Objective : We tried to investigate the PUVA effects on the proliferation and different,iation in cultured human keratinoc tes and melanocytes. Methods : We examined morphologic changes, and the number of the cultured human keratinocytes and melanoiytes and melanin contents in a control group and experimental group. (UVA group, 8-MOP group and PUVA group). i.e., UVA group was exposed to UVA at 60mJ, of. 8-MOP group was acJded at dose of 2 x 10 M to medium for 30 minutes. PUVA group was exposed to UVA at 60mJ, of after adding in 8 MOP at Zx 10 M for 30 minutes. Results : 1. Morphologic changes of cultured human keratinocytes and melanocytes. There were no significent changes between the control group and the experimental groups in keratinocytes and melanocytes after 24, 48 and 72 hours culture. The number and length of meianocyte dendrites showecl no significant differences between the groups after 24, 48 and 72 hours culture(p.>0.05). 2. Proliferation of cultured human keratinocytes and melanocytes 1) The number of keratinocytes in 8-MOP and PUVA groups decreased significantly more than in the control and LVA groups at 72nd hour after culture (p<0.01). 2) The number of melnocytes showed no differences between the groups at 72nd hour after culture (p>0.05). 3. Melanin contents in iultured human melanocytes The melanin contents increased significantly in the PUVA groups compared to that in the other groups at 72nd hour after culture (p<0.01). Conclusion : In culturel human keratinocytes, PUVA has no effect on the morphology and differentiation, hut inhibit proliferation. In cultured human melanocytes, PUVA has no effect on morphology and proliferation, but it increases the melanin contents. (Kor J Dermatol 1995;33(5): 886-894)